Characterization of the ADP-β-d-manno-heptose biosynthetic enzymes from two pathogenic Vibrio strains

Abstract ADP-activated β-d-manno-heptoses (ADP-β-d-manno-heptoses) are precursors for the biosynthesis of the inner core of lipopolysaccharide in Gram-negative bacteria. Recently, ADP-d-glycero-β-d-manno-heptose (ADP-d,d-manno-heptose) and its C-6′′ epimer, ADP-l-glycero-β-d-manno-heptose (ADP-l,d-manno-heptose), were identified as potent pathogen-associated molecular patterns (PAMPs) that can trigger robust innate immune responses. Although the production of ADP-d,d-manno-heptose has been studied in several different pathogenic Gram-negative bacteria, current knowledge of ADP-β-d-manno-heptose biosynthesis in Vibrio strains remains limited. Here, we characterized the biosynthetic enzymes of ADP-d,d-manno-heptose and the epimerase that converts it to ADP-l,d-manno-heptose from Vibrio cholerae (the causative agent of pandemic cholera) and Vibrio parahaemolyticus (non-cholera pathogen causing vibriosis with clinical manifestations of gastroenteritis and wound infections) in comparison with their isozymes from Escherichia coli. Moreover, we discovered that β-d-mannose 1-phosphate, but not α-d-mannose 1-phosphate, could be activated to its ADP form by the nucleotidyltransferase domains of bifunctional kinase/nucleotidyltransferases HldEVC (from V. cholerae) and HldEVP (from V. parahaemolyticus). Kinetic analyses of the nucleotidyltransferase domains of HldEVC and HldEVP together with the E. coli–derived HldEEC were thus carried out using β-d-mannose 1-phosphate as a mimic sugar substrate. Overall, our works suggest that V. cholerae and V. parahaemolyticus are capable of synthesizing ADP-β-d-manno-heptoses and lay a foundation for further physiological function explorations on manno-heptose metabolism in Vibrio strains. Key points • Vibrio strains adopt the same biosynthetic pathway as E. coli in synthesizing ADP-β-d-manno-heptoses. • HldEs from two Vibrio strains and E. coli could activate β- d -mannose 1-phosphate to ADP-β- d -mannose. • Comparable nucleotidyltransfer efficiencies were observed in the kinetic studies of HldEs. Supplementary Information The online version contains supplementary material available at 10.1007/s00253-024-13108-3.

The Vibrio genus is ubiquitously found in diverse aquatic and marine habitats.It comprises more than 12 species that can cause human infections.Human diseases caused by pathogenic Vibrio species are divided into two types: cholera and noncholera infections (Baker-Austin et al. 2018).V. cholerae is the causative agent of pandemic cholera, an acute watery diarrheal disease spreading via the fecal-oral and person-to-person transmission (Kanungo et al. 2022).Of the non-cholera pathogens, V. parahaemolyticus and Vibrio vulnificus are the representative species causing vibriosis including gastroenteritis, wound infection, and sepsis (Baker-Austin et al. 2018).Several virulence factors, e.g., enterotoxin, hemolysin, and LPS contribute considerably to the pathogenicity of Vibrio, thus being studied intensively (Zhang and Austin 2005).In V. cholerae, the LPS inner core is usually composed of at least three l-glycero-α-d-manno-heptoses, which are loaded by inverting glycosyltransferases employing ADP-l,d-manno-heptose as a sugar donor (Chatterjee and Chaudhuri 2003).However, little is known about the biosynthesis of ADP-activated β-d-mannoheptoses in Vibrio, let alone the influences of those ADP-β-dmanno-heptoses on Vibrio pathogenesis.
In this work, we analyzed the genomes of two pathogenic Vibrio strains, V. cholerae O1 2010EL-1786and V. parahaemolyticus CGMCC 1.1997 (ATCC 17802), and found that they possess the same set of ADP-β-d-manno-heptose biosynthetic enzymes as E. coli.Interestingly, though they display high protein sequence similarities with the E. coli-derived homologues (>60%), the predicted structural differences between the nucleotidyltransferase domains of the bifunctional HldEs aroused our interests to study their functions elaborately.The catalytic activities of those Vibrio enzymes were identified by in comparative investigations along with their isozymes from E. coli.Restricted by the availability of H1P, quantitative studies on the β-d-manno-heptose nucleotidyltransferases are difficult.Here, we show that the bifunctional HldEs from both Vibrio and E. coli could activate β-d-mannose 1-phosphate, but not α-d-mannose 1-phosphate, to form ADP-β-d-mannose.And the kinetic analyses of their nucleotidyltransferase domains were performed using β-d-mannose 1-phosphate, which revealed that they have comparable nucleotidyltransfer efficiencies.Taken together, we characterized the enzymes involved in ADP-β-d-manno-heptose synthesis in Vibrio strains and laid a foundation for further investigations on the influence of heptose metabolism on the virulence of Vibrio.

Bacterial strains and plasmids
Bacterial strains and plasmids used in this study are listed in Table S1.E. coli JM109 was used for general DNA cloning.E. coli BL21 (DE3) ΔgmhA ΔgmhB ΔhldE mutant was used for protein expression (Tang et al. 2022).LB broth and agar were used for the growth of E. coli strains at 37 °C.V. parahaemolyticus CGMCC 1.1997 was cultured at 37 °C in LB broth and used as the template for cloning the genes involving in ADP-β-d-manno-heptose biosynthesis from its genome (NCBI RefSeq assembly, GCF_001011015.1).ADP-β-d-manno-heptose biosynthetic genes from V. cholerae O1 2010EL-1786 (NCBI RefSeq assembly, GCF_000166455.1) were obtained by DNA synthesis.

DNA manipulation and sequence analysis
All PCR primers used in this study were synthesized by GENEWZ Co. (Suzhou, China) and listed in Table S2.DNA synthesis and sequencing were carried out in GENEWIZ Co. (Suzhou, China).PCRs were performed with PrimeSTAR HS DNA polymerase (Takara, Shiga, Japan) or Taq DNA polymerase (TransGene, Beijing, China) according to the manufacturers' instructions.A BLASTP search was used to predict protein functions (https:// blast.ncbi.nlm.nih.gov/ Blast.cgi).

Protein expression and purification
All the proteins were expressed in E. coli BL21 (DE3) ΔgmhA EC ΔgmhB EC ΔhldE EC mutant.A single transformant of the E. coli BL21 ΔgmhA EC ΔgmhB EC ΔhldE EC strain harboring a specific protein expression plasmid was inoculated into LB medium with 50 μg/mL kanamycin and cultured overnight at 37 °C, 220 rpm.Subsequently, the overnight culture was inoculated into LB with 50 μg/mL kanamycin at 1:100 dilution and incubated at 37 °C, 220 rpm until OD 600 reached 0.6.The expression of the candidate protein was then induced by adding isopropyl β-d-thiopyranogalactoside (IPTG) to a final concentration of 0.1 mM and cultured at 16 °C, 180 rpm for 18 h.
Protein purifications were carried out with Ni-NTA affinity column at 4 °C following the manufacturer's instructions.After harvesting the cell pellets by centrifugation, we resuspended them in binding buffer (20 mM Tris-HCl, 500 mM NaCl, 5 mM imidazole, 5% glycerol, pH 7.9) for sonication.Then, the cell debris was removed by centrifugation and the supernatant was loaded onto Ni-NTA affinity column preequilibrated with binding buffer.After being washed with washing buffer (20 mM Tris-HCl, 500 mM NaCl, 60 mM imidazole, 5% glycerol, pH 7.9) and elution buffer (20 mM Tris-HCl, 500 mM NaCl, 500 mM imidazole, 5% glycerol, pH 7.9) sequentially, the desired fractions were combined, desalted with PD-10 columns (GE Healthcare, USA), and concentrated by ultracentrifugation using an Amicon Ultra Centrifugal Filter device (Merck Millipore, USA; molecular mass cutoff of 10 kDa for the bifunctional HldEs and 3 kDa for the other proteins).The purified proteins were stored in 20 mM HEPES buffer (pH 8.0) with 200 mM NaCl and 10% glycerol at −80 °C (Li et al. 2021).Protein concentrations were measured by the Bradford assay (Bradford 1976).

Assays of the ADP-d,d-manno-heptose biosynthetic enzymes
The catalytic activities of ADP-d,d-manno-heptose biosynthetic enzymes were studied with the four-step assays using S7P and ATP as substrates.For the characterization of GmhA VC , GmhB VC , and HldE VC from V. cholerae O1 2010EL-1786, the reactions were performed in a 50-μL volume mixture containing 20 mM HEPES buffer (10% glycerol, 200 mM NaCl, pH 8.0), 2 mM MgCl 2 , 2 mM KCl, 2 mM ATP, 0.2 mM S7P, 5 μM GmhA VC , 5 μM GmhB VC , and 5 μM HldE VC at 30 °C for 2 h.And the well-studied combination of GmhA EC , GmhB EC , and HldE EC from E. coli was performed at the same conditions as a positive control.To check the catalytic activity of GmhA VC or GmhB VC or HldE VC , its corresponding isoenzyme in the combination of GmhA EC + GmhB EC + HldE EC was replaced by the one from V. cholerae O1 2010EL-1786.The assays of ADP-d,d-manno-heptose biosynthetic enzymes from V. parahaemolyticus CGMCC 1.1997 and the compensation experiments of their isoenzymes from E. coli were performed similarly as the enzymes from V. cholerae O1 2010EL-1786.All of the reactions were quenched by mixing vigorously with an equal volume of chloroform.After centrifugation, the chloroform layer was removed and 10 μL of aqueous sample was subjected to HPLC analysis (Tang et al. 2022).

Enzymatic assays of HldD VC , HldD VP , and HldD EC
The enzymatic reactions of C-6″ epimerases, HldD VC , HldD VP , and HldD EC , were performed at the same conditions and here takes HldD VC as example.The enzymatic assay of HldD VC was set in a 50-μL volume mixture containing 20 mM HEPES buffer (10% glycerol, 200 mM NaCl, pH 8.0), 2 mM NAD + , 0.2 mM ADP-d,d-manno-heptose, and 5 μM HldD VC at 30 °C for 1 h.The reaction was quenched by mixing vigorously with an equal volume of chloroform.After centrifugation, the chloroform layer was removed and 10 μL of aqueous sample was subjected to HPLC analysis.

Enzymatic assays of HldE using β-d-mannose 1-phosphate as a substrate
To test whether HldE could use β-d-mannose 1-phosphate as a substrate, the enzymatic reaction was carried out in a 50 μL volume mixture containing 20 mM HEPES buffer (10% glycerol, 200 mM NaCl, pH 8.0), 2 mM MgCl 2 , 2 mM ATP, 0.2 mM β-d-mannose 1-phosphate (Supplementary Scheme 1 and Fig. S1), and 5 μM HldE VC or HldE VP or HldE EC at 30 °C for 2 h.To test whether α-d-mannose 1-phosphate can be taken by HldE, the reactions were performed at the same conditions except that β-d-mannose 1-phosphate was replaced by α-d-mannose 1-phosphate.All the reactions were quenched by mixing vigorously with an equal volume of chloroform.After centrifugation, the chloroform layer was removed and 10 μL of aqueous sample was subjected to HPLC analysis.

Enzymatic assays of ADP-d-glycero-β-d-altro-heptose synthesis
To test whether HldE VC from V. cholerae O1 2010EL-1786 can synthesize ADP-d-glycero-β-d-altro-heptose, the enzymatic reactions were performed in a 50-μL volume mixture containing 20 mM HEPES buffer (10% glycerol, 200 mM NaCl, pH 8.0), 2 mM MgCl 2 , 2 mM KCl, 2 mM ATP, 0.2 mM S7P, 5 μM HygP, 5 μM GmhB VC , and 5 μM HldE VC at 30 °C for 2 h.The enzymatic assays of HldE VP from V. parahaemolyticus CGMCC 1.1997 were performed similarly except that HldE VC and GmhB VC were replaced by HldE VP and GmhB VP .The well-studied combination of HygP, GmhB EC , and HldE EC was performed as a positive control at the same conditions.All of the reactions were quenched by mixing vigorously with an equal volume of chloroform.After centrifugation, the chloroform layer was removed and 10 μL of aqueous sample was subjected to HPLC analysis.
HRMS was performed on an Agilent 1260 HPLC/6520 QTOF-MS instrument with an electrospray ionization source.NMR spectra were recorded at room temperature on a Bruker-500 NMR.

Colorimetric assay
A pyrophosphatase (PPase)-coupled colorimetric assay was developed for monitoring the nucleotidyltransfer activities of HldE VC , HldE VP , and HldE EC .Briefly, the enzymatic assay was carried out in a 50-μL volume mixture 96-well microtiter plate using ATP (0.2 mM) and β-d-mannose 1-phosphate (0.1 mM) as substrates.The PPase assays revealed that 0.1 U PPase could effectively catalyze the hydrolysis of 0.2 mM PPi at a wide temperature range (from 15 to 45 °C).Therefore, HldE was added together with 0.1 U PPase and the reaction mixture was incubated at the assay temperature for HldE.After 30 min, the reactions were terminated by adding the premixed malachite green and ammonium molybdate agent from the Malachite Green Phosphate Detection Kit (CST, MA, USA) at room temperature for 15 min and monitored by a microplate reader (Synergy H4, BioTek, USA) at 630 nm (Sha et al. 2012).The reactions with boiled HldEs were carried out as negative controls to adjust the interference by substrates and buffer components.

Kinetic studies
Using the developed colorimetric assay, the reaction conditions of HldE VC , HldE VP , and HldE EC were optimized.The optimal pH was determined by performing the enzyme reaction in 20 mM different buffers (citrate-sodium citrate buffer (pH 6.5), HEPES-NaOH (pH 7.0, 7.5, 8.0), Tris-HCl buffer (pH 8.5, 9.0), glycine-NaOH buffer (pH 9.5, 10.0)) at 30 °C.The optimal temperatures of the three enzymes were determined by incubating the reactions at 15 to 45 °C at their optimal pHs.The initial velocities were evaluated by performing the reactions at a different incubation time under the optimal pH and temperature.
The K m and k cat values of HldE VC , HldE VP , and HldE EC against β-d-mannose 1-phosphate and ATP were determined under their optimal pH and temperature.The K m values of the three enzymes against β-d-mannose 1-phosphate were obtained by performing the reactions with different concentrations of β-d-mannose 1-phosphate (5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, and 150 μM) and a saturation concentration of ATP (2 mM).The K m values of the three enzymes against ATP were obtained by performing the reactions with different concentrations of ATP (20,35,50,75,100,150,200,250,300,500,750, and 1000 μM) and a saturation concentration of β-d-mannose 1-phosphate (400 μM).Each parameter was measured in triplicate and the data were analyzed using Origin 2021.

Characterization of the Vibrio enzymes responsible for ADP-d,d-manno-heptose biosynthesis
V. cholerae O1 2010EL-1786 is a causative agent of lifethreating cholera isolated from a stool sample of one cholera patient (Reimer et al. 2011); V. parahaemolyticus CGMCC 1.1997 is a gastroenteritis causative strain from a patient suffering with Shirasu food poisoning (Daniel et al. 1999).In silico analysis of their genome sequences (NCBI RefSeq assemblies: GCF_000166455.1 (V.cholerae O1 2010EL-1786) and GCF_001011015.1 (V.parahaemolyticus CGMCC 1.1997)) revealed that both of the two strains contain the necessary genes responsible for ADPβ-d-manno-heptose biosynthesis.Sequence alignments revealed that the ADP-β-d-manno-heptose biosynthetic enzymes from the two Vibrio strains display more than 60% amino acid sequence identities with their homologues from E. coli (Table S3).AlphaFold prediction and subsequent structure analysis revealed that the overall structures of the isomerases, phosphatases, and ADP-l,d-manno-heptose C-6″ epimerases from both Vibrio strains are quite similar to their homologues from E. coli (Figs.S2 and S3), while prominent conformation differences that deviate severely from HldE EC were observed in both of the two HldEs from Vibrio, especially the nucleotidyltransferase domain of HldE VP (Fig. 1c), which aroused our interests to study their functions elaborately.We cloned the genes encoding β-dmanno-heptose isomerase (gmhA VP ), phosphatase (gmhB VP ), and bifunctional kinase/nucleotidyltransferase (hldE VP ) from V. parahaemolyticus CGMCC 1.1997.The genes encoding the same set of isozymes from V. cholerae O1 2010EL-1786, gmhA VC , gmhB VC , and hldE VC , were codon optimized and synthesized.The six genes were expressed as N-His 6 tagged proteins using a "clean" chassis strain E. coli BL21 (DE3) ΔgmhA ΔgmhB ΔhldE (Tang et al. 2022), in which the native β-d-manno-heptose biosynthetic genes were knocked out (Fig. 1c).
The catalytic activities of the enzymes responsible for ADP-d,d-manno-heptose synthesis in V. cholerae O1 2010EL-1786 were investigated by incubating GmhA VC , GmhB VC , and HldE VC with S7P and ATP.A product shared the same retention time with ADP-d,d-manno-heptose was generated and was further identified by HPLC co-injection with the authentic standard of ADP-d,d-manno-heptose.We also tested the hybrid assays by replacing the well-characterized isomerase, phosphatase, or kinase/nucleotidyltransferase in the combination of GmhA EC + GmhB EC + HldE EC , which can synthesize ADP-d,d-manno-heptose efficiently, with the corresponding isozyme from V. cholerae O1 2010EL-1786, and all the combinations converted S7P to ADP-d,d-mannoheptose with comparable efficiencies (Fig. 1d).
When the heptose synthetic enzymes from V. parahaemolyticus CGMCC 1.1997 were incubated with S7P and ATP, ADP-d,d-manno-heptose was also generated in the GmhA VP + GmhB VP + HldE VP combination, but not in the control reaction with boiled HldE VP .And the enzymes from V. parahaemolyticus CGMCC 1.1997 could also functionally replace their isozymes in the GmhA EC + GmhB EC + HldE EC combination (Fig. 1d).HPLC analyses showed that about 72 μM, 64 μM, and 80 μM of S7P (200 μM) were converted to ADP-d,d-manno-heptose by HldE VC , HldE VP , and HldE EC together with its cognate GmhA and GmhB, respectively, at 30 °C, pH 8.0 (20 mM HEPES) for 2 h.The comparable production of ADP-d,d-manno-heptose could be explained by docking analysis of ATP in the binding pockets of the nucleotidyltransferase domains of HldEs, which showed that despite the differences in tertiary structures of the HldEs, they may adopt similar "horseshoe" binding model of ATP to shape ADP-d,d-manno-heptose effectively (Fig. S4).Collectively, these results confirmed that GmhA VC and GmhA VP are S7P isomerases, GmhB VC and GmhB VP are HBP phosphatases, and HldE VC and HldE VP are bifunctional proteins with H7P kinase and H1P nucleotidyltransferase activities.
To verify the function of HldD VC , it was incubated with ADP-d,d-manno-heptose and NAD + .HPLC analysis showed that ADP-d,d-manno-heptose was converted to a compound having the same retention time as the authentic standard of ADP-l,d-manno-heptose, and it was further verified by HRMS analysis (m/z 618.0847 for [M-H] − , C 17 H 27 N 5 O 16 P 2 , cacld 618.0855) (Fig. S5).HldD VP could also convert ADP-d,d-manno-heptose to ADP-l,d-mannoheptose under the same assay conditions.Both of the two ADP-d,d-manno-heptose C-6″ epimerase from Vibrio species displayed comparable catalytic efficiencies with the E. coli epimerase HldD EC , which was used as a positive control (Fig. 2c).Taken together, we showed that both V. cholerae O1 2010EL-1786 and V. parahaemolyticus CGMCC 1.1997 possess the complete set of β-d-manno-heptose biosynthetic enzymes for synthesizing ADP-l,d-manno-heptose from S7P, implying that l,d-manno-heptose is widely distributed in the LPSs of different Vibrio strains.

Synthesis of ADP-β-d-mannose by the nucleotidyltransferase domain of HldE
A study on HldE EC suggested that it can convert mannose to ADP-β-d-mannose in the presence of ATP, indicating that the kinase domain of HldE EC is able to add a phosphate group onto the anomeric carbon of mannose to form β-d-mannose 1-phosphate, which is then converted to ADP-β-d-mannose by the nucleotidyltransferase domain of HldE EC (Morrison and Tanner 2007).To verify the substrate promiscuity of the nucleotidyltransferase domain of HldE EC , we chemically synthesized β-dmannose 1-phosphate as described (Supplementary Scheme 1 and Fig. S1).When HldE EC was incubated with ATP and the mimic sugar substrate, β-d-mannose 1-phosphate, or α-dmannose 1-phosphate (commercially available), a new peak was observed only in the assay of β-d-mannose 1-phosphate, while not in the assay using α-d-mannose 1-phosphate or the negative control using boiled HldE EC (Fig. 3b; Fig. S6).Subsequently, the product was prepared by a large-scale enzymatic synthesis and confirmed to be ADP-β-d-mannose by careful analyses of its high-resolution mass spectrometry and nuclear magnetic resonance data (Fig. 3c).The β-configuration of the anomeric carbon of mannose was assigned by the NOE correlations of H-1″ with H-3″ and H-5″ (Fig. S7).We also tested the catalytic activities of HldE VC and HldE VP toward β-d-mannose 1-phosphate (200 μM) and α-d-mannose 1-phosphate.Both of the two enzymes could only take β-d-mannose 1-phosphate, activate it into ADP-β-d-mannose as HldE EC , and comparable yields of ADP-β-d-mannose (76 μM for HldE VC , 68 μM for HldE VP , and 76 μM for HldE EC ) were detected at 30 °C, pH 8.0 (20 mM HEPES), 2 h (Fig. 3a, b; Fig. S6).The results suggested that the nucleotidyltransferase domain of HldE possesses a certain level of promiscuity toward the size of the sugar substrates, but it has a stringent specificity on the anomeric configuration of sugar 1-phosphate.

Kinetics of the nucleotidyltransferase domains of HldE VC , HldE VP , and HldE EC
As aforementioned, ADP-β-d-manno-heptoses are important components of LPS.However, due to the difficulties in preparing H1P, the catalytic efficiency of the nucleotidyltransferase domain of HldE, which is responsible for activating H1P into ADP-d,d-manno-heptose, has not been investigated kinetically yet.If the real substrate of a nucleotidyltransferase is unavailable, its mimic substrate can be used instead for kinetic studies (Kim et al. 2020;Kim et al. 2021).With β-d-mannose 1-phosphate in hand, we collected the kinetic parameters of HldE using it and ATP as substrates.
The nucleotidyltransferase domain of HldE cleaves an ATP into an AMP and one molecule of pyrophosphoric acid (PPi).The AMP is employed to activate β-d-pyranose 1-phosphate into ADP-β-d-pyranose, and the PPi can be hydrolyzed into inorganic phosphate (Pi) by a PPase and measured conveniently by a colorimetric assay as shown in Fig. 4a.Pi reacts with the premixed malachite green and ammonium molybdate agent under acidic condition to generate a malachite green complex, and the signal can be easily read at 630 nm.We developed a stable detection process by controlling the reaction and the detection conditions to minimize the background influence.Then, the reaction conditions of HldE VC and HldE VP as well as HldE EC were optimized by measuring the Pi generation during the process.It was showed that the three enzymes reached their maximal activities at 30 °C, and the optimum pHs of HldE VC , HldE VP , and HldE EC were 8.5, 8.0, and 7.5, respectively (Figs.S8 and S9).Subsequently, steady-state kinetic studies were carried out under the optimized conditions.The K m and k cat values against both substrates, β-d-mannose 1-phosphate and ATP, were collected and summarized in Fig. 4b.HldE VC and HldE VP displayed comparable K m and k cat values.HldE EC from E. coli exhibited slightly higher affinities to β-d-mannose 1-phosphate and ATP substrates than HldEs from Vibrio species, while it had comparable turnover numbers (Fig. 4b; Fig. S10).

Discussion
Vibrio genus contains several important pathogenic species (e.g., V. cholerae and V. parahaemolyticus) that can cause human diseases.Many people around the world are suffering waterborne and foodborne vibriosis with symptomatic entities such as watery diarrhea, stomach cramping, nausea, vomiting, fever, and chills (Dutta et al. 2021).ADP-β-d-mannoheptoses participate in the assembly of LPS, a virulence factor of Vibrio infections (Qadri et al. 2003;Chatterjee and Chaudhuri 2003), and are also powerful agonists that could trigger NF-κB-mediated innate immune responses (Janeway Jr. and Medzhitov 2002;Zhou et al. 2018).However, little is known about the formation mechanism of ADP-β-d-mannoheptoses in Vibrio species or the influences of those ADPsugars on their pathogenesis.In this work, we characterized the manno-heptose biosynthetic enzymes from two Vibrio strains and proposed that Vibrio adopts the same biosynthetic pathway as E. coli to synthesize ADP-d,d-mannoheptose and then ADP-l,d-manno-heptose using S7P as a precursor.In this process, S7P goes through a four-step reaction relay including isomerization, phosphorylation at C-1″, dephosphorylation at C-7″, and nucleotide activation to form ADP-d,d-manno-heptose. ADP-d,d-manno-heptose is further epimerized at C-6″ to generate ADP-l,d-mannoheptose, which is then loaded to lipid A to assemble the inner  (Whitfield and Trent 2014).To date, a number of different H1P nucleotidyltransferases have been studied enzymatically (Kneidinger et al. 2002;Park et al. 2018;Tang et al. 2022).However, none of them, even the well-characterized HldE EC from E. coli, has been investigated kinetically, mainly due to the difficulties in obtaining the sugar substrate, H1P.We chemically synthesized a mimic substrate, β-d-mannose 1-phosphate, and verified that all of the HldEs from E. coli and Vibrio strains could take it and catalyze the conversion of β-d-mannose 1-phosphate to ADP-β-d-mannose with considerable efficiencies.Thus, kinetic analyses of the three HldEs were performed using the synthesized β-d-mannose 1-phosphate as the sugar donor and ATP as the acceptor.The results showed that the two Vibrio enzymes, HldE VC and HldE VP , exhibited comparable nucleotidyltransfer efficiencies, while HldE EC from E. coli outperformed a little bit on affinities of both substrates.Actually, if the real substrate of a nucleotidyltransferase is unavailable, its mimic substrate can be used instead for kinetic studies (Kim et al. 2020;Kim et al. 2021).While this approach may not capture the natural properties of the enzyme, it can still provide us valuable information and enhance our understanding to the targeted enzyme.In addition, our previous works revealed that HldE EC is able to tolerate the 3-epimer of H1P and activate d-glycero-β-d-altro-heptose 1-phosphate into its ADP form (Tang et al. 2018) and the Vibrio strain-derived HldE VC and HldE VP also possess similar abilities to synthesize ADPd-glycero-β-d-altro-heptose (Fig. S11).Taken collectively, HldEs exhibit a certain level of sugar substrate flexibilities and can take not only different β-d-heptose 1-phosphate but also β-d-mannose 1-phosphate (Adekoya et al. 2018).They may be developed as potent catalysts for the synthesis of various ADP-β-d-sugars that are not easily synthesized by chemical methods.
In summary, we characterized the biosynthetic enzymes of ADP-β-d-manno-heptoses from two pathogenic Vibrio strains, which suggested that Vibrio strains adopt the same biosynthetic pathway as E. coli in synthesizing ADP-β-dmanno-heptoses.Moreover, we showed that the two HldEs from Vibrio could activate β-d-mannose 1-phosphate to its ADP form as well as HldE EC from E. coli and studied the kinetics of the nucleotidyltransferase domains of these three HldEs using this mimic substrate.All of our works enhance our understanding of ADP-β-d-manno-heptose biosynthesis in Vibrio strains and laid a foundation for the following studies on heptose metabolism in Vibrio and its influences on Vibrio pathogenesis.

Fig. 3
Fig. 3 Conversion of β-dmannose 1-phosphate to ADPβ-d-mannose.a A proposed reaction to form ADP-β-dmannose. b HPLC profiles of the enzymatic assays catalyzed by the nucleotidyltransferase domains of HldEs using β-dmannose 1-phosphate and ATP as substrates.The detection wavelength was set as 254 nm.c 1 H NMR (500 MHz) and 13 C NMR (125 MHz) data for ADPβ-d-mannose in D 2 O

Fig. 4
Fig. 4 Determination of the kinetics of the nucleotidyltransferase domains of HldEs.a A scheme of the colorimetric assay for detecting the formed PPi group that is generated by the nucleotidyltrans-